A biological sample encapsulation and transfer method suitable for solid nuclear magnetic resonance research and a matching centrifugal device

Abstract

The present invention discloses a biological sample encapsulation and transfer method suitable for solid nuclear magnetic resonance research and a matching centrifugal device, which belongs to the field of solid nuclear magnetic detection technology. The transfer method is to transfer the sample to be tested into the nuclear magnetic rotor by a one-time centrifugation, and is used in conjunction with the centrifugal device of the present invention. The centrifugal device includes a detachably connected upper device and a lower device, the upper device is provided with a flow guide channel that runs through the upper device from top to bottom, and the lower device is provided with a tube placement groove for placing the nuclear magnetic rotor. Among them, the head end of the flow guide channel is used for sample introduction, and the tail end is used to communicate with the tube placement groove. The one-time centrifugal transfer method of the present invention can avoid excessive human intervention. The centrifugal device of the present invention can effectively transfer the sample to the nuclear magnetic rotor, and make the sample uniformly distributed in the nuclear magnetic rotor, fully and evenly fill the nuclear magnetic rotor space, and avoid the sample center of gravity shift during the nuclear magnetic experiment, resulting in poor test results or even experimental operation risks.

Description

Technical Field

[0001] The invention relates to the technical field of nuclear magnetic resonance detection, in particular to a biological sample encapsulation and transfer method suitable for solid nuclear magnetic resonance research and a matching centrifugal device. Background Art

[0002] Solid-state NMR technology is one of the few important means to obtain atomic-scale information of biological macromolecules under near-physiological conditions. In recent years, solid-state NMR technology has made great progress both in hardware equipment and in principle and method, and biological solid-state NMR experiments have become easier to implement. The premise of obtaining structural or dynamic information of biological macromolecules such as proteins using solid-state NMR is to obtain their high-resolution NMR spectra. Unlike the inherent high resolution of solution samples, solid or semi-solid samples lack rapid random motion, resulting in the inability to offset various anisotropic interactions such as chemical shifts and dipole interactions, causing serious broadening effects on the spectrum, and generally unable to obtain high-resolution NMR spectra.

[0003] In solid-state nuclear magnetic resonance, an effective method to eliminate this anisotropic interaction is the magic angle rotation technique of the sample, which is to rapidly rotate the sample to be tested at a specific angle of 54.7° to the main magnetic field. When the rotation speed is fast enough to exceed the anisotropic interaction, the broadening effect of the spectrum is suppressed, thereby obtaining a high-resolution spectrum.

[0004] In specific experiments, we often need to encapsulate the sample in a ceramic cylindrical tube (called a nuclear magnetic resonance rotor). The cap is made of high-quality plastic and has some impellers on it. The rotor can be driven to rotate at high speed by compressed air. According to the different needs of the experiment, the current commercial rotors also have different sizes and models, with outer diameters ranging from 0.7mm to 7mm and lengths generally around 15mm. The maximum speed that can be achieved by rotors of different sizes in experiments ranges from several thousand Hz to hundreds of thousands of Hz. Due to the small size of the rotor and the high speed in the experiment, the accuracy of various operations in the experiment is very high, so it is not easy to load the sample into the rotor in the nuclear magnetic resonance experiment. The nuclear magnetic signal is very sensitive to the conformation and environment of the sample. Slight differences in conformation or environment will cause signal deviation. For a macroscopic sample, this local heterogeneity will directly lead to a decrease in signal resolution on the spectrum. Therefore, it is necessary to be extra careful when transferring and loading the purified biological sample into the tube, and the integrity and water content of the sample should be maintained as much as possible. This is not only a guarantee of spectral quality, but also a requirement for biological samples to maintain their biological functionality. In biological solid NMR, the sample is usually loaded by ultracentrifuging the purified sample solution or flocculent aggregate to obtain a gel-like high-density sample, which is then transferred to the rotor by centrifugation or manually using a small spoon. Manual transfer is time-consuming and labor-intensive, and inevitably causes some sample loss. Summary of the invention

[0005] In view of the deficiencies of the prior art described above, the present application provides a biological sample packaging and transfer method suitable for solid nuclear magnetic resonance research and a matching centrifugal device. The transfer method is to transfer the sample to be tested into a nuclear magnetic rotor by a one-time centrifugation, and use it in conjunction with the centrifugal device of the present invention, which can avoid excessive human intervention. The centrifugal device can effectively transfer the sample to the nuclear magnetic sample tube, and make the sample evenly distributed in the nuclear magnetic rotor, fully and evenly fill the nuclear magnetic rotor space, and avoid the center of gravity of the sample from shifting during the nuclear magnetic experiment, resulting in poor test results or even experimental operation risks.

[0006] The first aspect of the present application provides a biological solid nuclear magnetic sample centrifuge device, which includes an upper part and a lower part of the device, and the bottom of the upper part of the device is detachably connected to the top of the lower part of the device; the upper part of the device is provided with a guide channel, and the guide channel runs through the upper part of the device from top to bottom; the lower part of the device is provided with a tube placement groove for placing a nuclear magnetic rotor; wherein the head end of the guide channel is used for sample introduction, and the tail end is used to communicate with the tube placement groove.

[0007] In some embodiments of the present invention, a flow converging portion and a flow guiding portion are provided on the upper portion of the device, and the flow converging portion and the flow guiding portion are provided with a flow converging cavity and a flow guiding cavity respectively, and the flow converging cavity and the flow guiding cavity are connected to form the flow guiding channel;

[0008] The inner diameter of the converging cavity is larger than the inner diameter of the diverting cavity.

[0009] In some embodiments of the present invention, the inner diameter of the guide cavity decreases from top to bottom.

[0010] In some embodiments of the present invention, the flow converging portion and the flow guiding portion are both hollow to form the flow converging cavity and the flow guiding cavity;

[0011] And / or, the converging portion is used to place the ultracentrifuge sample tube, and the shape of the converging portion is adapted to the shape of the sample outlet of the ultracentrifuge sample tube.

[0012] In some embodiments of the present invention, the bottom of the upper portion of the device is plug-connected or screw-connected to the top of the lower portion of the device.

[0013] In some embodiments of the present invention, a boss is provided at the bottom of the upper portion of the device, a groove matching the boss is provided at the top of the lower portion of the device, and the groove is located at the top of the tube placement groove and communicated with the tube placement groove;

[0014] Wherein, the guide channel passes through the boss and the guide channel coincides with the center line of the tube placement groove.

[0015] In some embodiments of the present invention, the boss includes a connecting section and a hollow extension section, the connecting section is adapted to the shape of the groove, and the outer diameter of the hollow extension section is smaller than the inner diameter of the tube placement groove;

[0016] And / or, a sealing ring is provided on the outer side of the boss or the inner side of the groove; or, the boss is threadedly connected to the groove.

[0017] In some embodiments of the present invention, the difference between the length of the connecting section and the depth of the groove is 0 to 1 mm;

[0018] And / or, the length of the hollow extension section is 1 to 3 mm.

[0019] In some embodiments of the present invention, the length of the upper part 1 of the device is 20 to 40 mm, preferably 25 to 35 mm; the length of the lower part of the device is 20 to 40 mm, preferably 25 to 35 mm; the device adopts a reasonable length, which can shorten the ultracentrifugation time, avoid the operational risks that may be caused by long-term centrifugation, and maintain the biological functionality of the sample to be tested.

[0020] In some embodiments of the present invention, the centrifugal device is made of a material that meets the requirements of high-speed centrifugation, such as a plastic material;

[0021] And / or, the upper portion of the device is cylindrical, the lower portion of the device is cylindrical with a hemispherical bottom, and the outer diameters of the upper portion and the lower portion of the device are adapted to the inner diameter of the centrifuge tube;

[0022] And / or, the centrifugal device is made by 3D printing.

[0023] In some embodiments of the present invention, the centrifugal device is made of materials such as polytetrafluoroethylene, polyetheretherketone, polychlorotrifluoroethylene, polyoxymethylene or polyimide.

[0024] The second aspect of the present invention is to provide a biological sample packaging and transfer method suitable for solid-state nuclear magnetic resonance research, wherein the transfer method is to transfer the sample in the sample tube to the nuclear magnetic rotor through a one-time centrifugation.

[0025] In some embodiments of the present invention, the transfer method is completed by using the centrifugal device, ultracentrifuge sample tube, nuclear magnetic rotor and centrifuge tube as described in any of the above items, and comprises the following steps:

[0026] S1. Enrich the sample to be tested into the ultracentrifuge sample tube. Generally, the sample to be tested is enriched into the sample tube by centrifugation. The ultracentrifuge sample tube generally uses a conventional centrifuge tube (volume of 1-3 ml). The centrifuge rotor is placed horizontally, the centrifuge speed is 50000-60000 rpm, the working temperature is 20-28 ° C, the centrifugation time is 30-120 min, and the mass of the sample to be tested is 5-20 mg. More specifically, after centrifugation, the supernatant of the sample tube is removed, and the water content is maintained at 20%-50%. In addition, absorbent paper can be used to remove droplets hanging on the wall of the sample tube. Maintaining a certain range of water content is conducive to sample transfer and maintaining sample uniformity, thereby obtaining a high-resolution spectrum of a high-resolution signal, and maintaining a certain water content is conducive to maintaining the biological functionality of the biological sample.

[0027] S2, placing the nuclear magnetic rotor into the lower part of the device, and then sequentially assembling the lower part of the device, the upper part of the device and the ultracentrifuge sample tube into the centrifuge tube;

[0028] S3, transfer the sample to be tested in the ultracentrifuge sample tube to the nuclear magnetic rotor through ultracentrifugation. The entire device needs to be weighed and balanced. The working conditions of the ultracentrifugation are: the centrifuge rotor is placed horizontally, the centrifuge speed is 15000-25000rpm, the working temperature is 3-5°C, and the centrifugation time is 10-30min.

[0029] By using the centrifugal device of the present invention in combination with the transfer method and corresponding transfer working parameters, the sample can be evenly distributed in the nuclear magnetic rotor, fully and evenly filling the nuclear magnetic rotor space, and avoiding the deviation of the center of gravity of the sample during the nuclear magnetic experiment.

[0030] The present invention provides a biological sample encapsulation and transfer method and a centrifugal device suitable for solid nuclear magnetic resonance research, which have the following beneficial effects:

[0031] 1) The one-time centrifugal transfer method is adopted, which reduces the number of transfers compared with the existing technology, avoids human intervention and avoids sample loss during the transfer process.

[0032] 2) The centrifugal device of the present invention is applied to the one-time centrifugal loading of solid samples, avoiding excessive human intervention; and the centrifugal device can effectively transfer the sample to the nuclear magnetic rotor, and make the sample evenly distributed in the nuclear magnetic rotor, fully and evenly fill the nuclear magnetic rotor space, and avoid the deviation of the center of gravity of the sample during the nuclear magnetic experiment.

[0033] 3) The centrifugal device of the present invention has high adaptability and can be adapted to commonly used centrifuges, rotors and centrifuge tubes. It is flexible and quick to use, has a short centrifugation time, and ensures the integrity and water content of the sample.

[0034] 4) The centrifugal device of the present invention has few parts and is easy to assemble. It does not rely on screws or glue. Through the cooperation between the connecting section and the groove and the centrifugal force, the parts will not move left and right or up and down, reducing the chance of sample leakage.

[0035] 5) The upper and lower parts of the device are detachably connected and can be designed in different models respectively, with high adaptability. For example, different models of lower parts of the device can be set according to different types of nuclear magnetic rotors, and the same upper part of the device can be adapted and replaced as needed.

[0036] 6) The device is easy to prepare and can be prepared by micro machine tools, CNC machine tools or 3D printing. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the split structure of the upper part and the lower part of the centrifugal device of the present invention;

[0038] Figure 2 A schematic front view of the upper portion of the centrifugal device of the present invention;

[0039] Figure 3 A schematic top view of the upper portion of the centrifugal device of the present invention;

[0040] Figure 4 It is a schematic diagram of the AA cross section of the upper part of the centrifugal device of the present invention;

[0041] Figure 5 A schematic front view of the lower portion of the centrifugal device of the present invention;

[0042] Figure 6 A schematic top view of the lower portion of the centrifugal device of the present invention;

[0043] Figure 7 It is a BB cross-sectional schematic diagram of the lower part of the centrifugal device of the present invention;

[0044] Figure 8 is a flow chart of the use of the centrifugal device of the present invention;

[0045] Fig. 9 One-dimensional human RIP3 fibers 1 H spectrum;

[0046] Fig.10 Human RIP3 fibers 13 C- 13 C 2D correlation spectrum;

[0047] Fig.11 The mouse RIPK3 fiber obtained in Example 5 13 C- 13 C 2D correlation spectrum;

[0048] Fig.12 is the mouse RIPK3 fiber obtained in the comparative example 13 C- 13 C two-dimensional correlation spectrum.

[0049] Numbers in the figure:

[0050] 1. The upper part of the device;

[0051] 11. Convergence Department;

[0052] 111, will flow cavity;

[0053] 12. Diversion part;

[0054] 121, diversion cavity;

[0055] 13. Boss;

[0056] 131, connecting section;

[0057] 132, hollow extension section;

[0058] 2. Lower part of the device;

[0059] 21. Catheter placement slot;

[0060] 22. Groove;

[0061] 100. Ultracentrifuge sample tube;

[0062] 200, nuclear magnetic rotor;

[0063] 300. Centrifuge tube. DETAILED DESCRIPTION

[0064] When the biological solid samples used for nuclear magnetic research are colloidal or pasty, it is more difficult to load the solid nuclear magnetic sample. At the same time, the preparation of biological solid nuclear magnetic samples is also more difficult and costly (>1,000 yuan / mg). How to minimize the loss during the sample transfer is also a problem that needs to be fully considered during the sample loading process. In order to reduce the number of transfers and human intervention, the applicant considers using a centrifugal device to quickly centrifuge the wet sample into the rotor at one time, and develops a matching biological solid nuclear magnetic sample centrifugal device and a fast and efficient sample loading process. The centrifugal device is combined with a centrifuge commonly used in biological laboratories, a matching rotor and a centrifuge tube, and can effectively (close to 100%) transfer samples to the nuclear magnetic rotor. The sample can fully and evenly fill the space in the nuclear magnetic rotor, effectively avoiding the offset of the sample center of gravity during the nuclear magnetic experiment. On this basis, the present invention is completed.

[0065] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0066] In the description of the present invention, it should be noted that the orientations or positional relationships indicated by terms such as “upper”, “lower”, “bottom”, “top”, “head end”, “tail end”, “two ends”, etc. are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the present invention.

[0067] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installation", "setting / provided with", "connection", etc. should be understood in a broad sense. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0068] Example 1

[0069] See also Figure 1-7 The centrifugal device comprises an upper device part 1 and a lower device part 2, and the bottom of the upper device part 1 is detachably connected to the top of the lower device part 2. Figure 1 As shown, the upper part 1 of the device is provided with a flow channel that runs through the upper part 1 from top to bottom. The lower part 2 of the device is also provided with a tube placement groove 21 for placing the nuclear magnetic rotor 200. The head end of the flow channel is used for sample introduction, and the tail end is used to communicate with the tube placement groove 21.

[0070] The centrifugal device is used in conjunction with an existing or customized nuclear magnetic rotor 200, an ultracentrifuge sample tube 100, and a centrifuge tube 300, so the shapes and sizes of the upper device 1, the lower device 2, and the tube slot 21 of the lower device 2 are designed according to the shapes and sizes of the nuclear magnetic rotor 200, the ultracentrifuge sample tube 100, and the centrifuge tube 300. Figure 8As shown, a centrifuge tube 300 adapted to the existing centrifuge equipment is used, so the upper part 1 of the device is cylindrical, the lower part 2 of the device is cylindrical with a hemispherical bottom, and the outer diameters of the upper part 1 and the lower part 2 of the device are adapted to the inner diameter of the centrifuge tube 300. That is, the length and outer diameter of the upper part 1 and the lower part 2 of the device are designed according to the length and inner diameter of the centrifuge tube 300. Preferably, the upper part 1 and the lower part 2 of the device are well fitted with the inner tube wall of the centrifuge tube 300, and at the same time, the length of the device and the length of the ultracentrifuge sample tube 100 are ensured to be less than the length of the centrifuge tube 300. The width and depth of the tube slot 21 are designed according to the size of the nuclear magnetic rotor 200. Generally, the length of the upper part 1 of the device is 20 to 40 mm, and the length of the lower part of the device is 20 to 40 mm. The use of a reasonable length of the device can shorten the time of ultracentrifugation, avoid the operational risks that may be caused by long-term centrifugation, and maintain the biological functionality of the sample to be tested.

[0071] The top size of the guide channel is designed according to the size of the ultracentrifuge sample tube 100 used, and the bottom size of the guide channel is designed according to the caliber of the entrance of the nuclear magnetic rotor 200. Generally, the inner diameter of the guide channel gradually decreases from top to bottom. Its purpose is to provide a channel for the sample in the ultracentrifuge sample tube 100 to be introduced into the nuclear magnetic rotor 200 during use.

[0072] In a specific embodiment, the upper part 1 of the device is provided with a converging portion 11 and a guiding portion 12, and the converging portion 11 and the guiding portion 12 are provided with a converging cavity 111 and a guiding cavity 121 respectively, and the converging cavity 111 and the guiding cavity 121 are connected to form a guiding channel. Among them, the inner diameter of the converging cavity 111 is larger than the inner diameter of the guiding cavity 121. The converging portion 11 is used to place the ultracentrifuge sample tube 100, and the shape of the converging portion 11 is adapted to the shape of the sample outlet of the ultracentrifuge sample tube 100, that is, the converging portion 11 is used to communicate with the outlet of the ultracentrifuge sample tube 100, so that the viscous sample in the ultracentrifuge sample tube 100 is introduced into the converging portion 11 during the centrifugation process, and then enters the rotor 200 placed in the tube placement groove 21 through the converging portion 11 and the guiding portion 12.

[0073] In another specific embodiment, the inner diameter of the flow guiding cavity 121 decreases from top to bottom to play a flow guiding role.

[0074] In a preferred embodiment, the converging portion 11 and the guiding portion 12 are both hollow to form a converging cavity 111 and a guiding cavity 121. Specifically, a method for forming the converging cavity 111 and the guiding cavity 121 is provided: a cavity (groove) is directly opened in the upper portion 1 of the device to form a guiding channel including the converging cavity 111 and the guiding cavity 121. Of course, the upper portion 1 of the device and the lower portion 2 of the device can also be directly made by 3D printing.

[0075] In a preferred embodiment, a detachable connection mode is provided between the bottom of the upper device part 1 and the top of the lower device part 2, such as plug-in or screw-on connection.

[0076] In a specific embodiment, if Figure 2 , Figure 4 , Figure 5 or Figure 7 As shown, a boss 13 is provided at the bottom of the upper part 1 of the device, and a groove 22 adapted to the boss 13 is provided at the top of the lower part 2 of the device. The groove 22 is located at the top of the tube placement groove 21 and is connected to the tube placement groove 21. The purpose is to make the upper part 1 of the device and the lower part 2 of the device tightly combined without any up-down or left-right translation through the plug-in cooperation of the boss 13 and the groove 22. In addition, the guide channel runs through the boss 13, and the center line of the guide channel coincides with the center line of the tube placement groove 21. Specifically, the boss 13 is cylindrical and is integrally formed with the upper part 1 of the device. The centrifugal device has few parts and is simple to assemble. It does not require screwing or gluing. Through the cooperation between the connecting section and the groove and the centrifugal force, the parts will not move left-right or up-down, thereby reducing the chance of sample leakage.

[0077] In a specific embodiment, a sealing ring is disposed at the connection between the boss 13 and the groove 22, for example, an O-ring is disposed on the outer side of the boss 13 or the inner side of the groove 22, to improve the sealing performance and reduce liquid leakage.

[0078] In a specific embodiment, the boss 13 is threadedly connected to the groove 22. Specifically, an outer side of the boss 13 is provided with an external thread, and an inner side of the groove 22 is provided with an internal thread. The threaded connection improves stability and reduces leakage.

[0079] In a preferred embodiment, if Figure 2 and Figure 4 As shown, the boss 13 includes a connecting section 131 and a hollow extension section 132. The connecting section 131 is adapted to the shape of the groove 22, and the outer diameter of the hollow extension section 132 is smaller than the inner diameter of the tube placement groove 21. The design of the hollow extension section 132 can play a better role in guiding the flow, so that the sample can fully and evenly fill the space in the rotor during the centrifugation process.

[0080] In a preferred embodiment, the difference between the length of the connecting section 131 and the depth of the groove 22 is 0 to 1 mm, preferably 0.1 to 0.3 mm, to ensure that the length of the connecting section 131 is slightly greater than the depth of the groove 22, which not only provides an assembly position for the sealing ring, but also ensures that during the centrifugation process, the overall force acts on the lower part 2 of the device, and will not cause pressure on the nuclear magnetic rotor 200 located in the groove 22, thereby avoiding damage to the nuclear magnetic rotor 200.

[0081] In a specific embodiment, the centrifugal device is made of a material that meets the requirements of high-speed centrifugation, that is, it needs to have a certain compressive resistance and not deform under high-speed centrifugation. It can be made of engineering plastics, such as polytetrafluoroethylene (PTEE), polyetheretherketone (PEEK), polychlorotrifluoroethylene (Kel-F), polyoxymethylene (delrin) or polyimide (vespel). Different materials have different strengths and mechanical properties, and the corresponding materials can be used according to the required centrifugal force.

[0082] Example 2

[0083] A centrifuge device with specific parameters is provided. The design is based on the most commonly used Bruker rotor with an outer diameter of 3.2 mm (generally 15 mm in length) and a 13.2 ml centrifuge tube (polypropylene, open-top thin wall) in solid-state nuclear magnetic resonance experiments. The centrifuge device is made of polytetrafluoroethylene (PTFE) material.

[0084] like Figure 2 As shown, the flow channel formed by the flow chamber 111 and the flow chamber 121 of the upper part 1 of the device is funnel-shaped, and a protruding boss 13 is provided at the bottom of the upper part 1 of the device, wherein the boss 13 also specifically includes a connecting section 131 and a hollow extension section 132, and a protruding secondary step is formed at the bottom of the upper part 1 of the device. The lower part 2 of the device is provided with a pipe placement groove 21 for placing the rotor, and a groove 22 is also provided on the upper side of the pipe placement groove 21. The matching relationship between the connecting end 131 and the groove 22 allows the upper part 1 of the device and the lower part 2 of the device to fit tightly, and also allows the hollow extension section 132 to enter the nuclear magnetic rotor 200 located in the pipe placement groove 21.

[0085] The dimensions of the above-mentioned parts are as follows: the length of the upper part 1 of the device is 30 mm, and the outer diameter is 13.5 mm; the inner diameter of the confluence cavity 111 is 11 mm, and the length is 8 mm; the length of the diversion cavity 121 is 25.5 mm, and the inner diameter decreases from top to bottom, and is 2 to 11 mm; the outer diameter of the connecting section 131 of the boss 13 is 6.5 mm, and the length is 3.5 mm; the length of the hollow extension section 132 of the boss 13 is 2 mm, and the outer diameter is 2 mm. The length of the lower part 2 of the device is 32.75 mm, and the outer diameter is 13.5 mm; the depth of the groove 22 is 3.3 mm, and the inner diameter is 6.6 mm; the inner diameter of the tube placement groove 21 is 3.2 mm, and the depth is 15.5 mm.

[0086] Example 3

[0087] A biological sample encapsulation and transfer method suitable for solid-state nuclear magnetic resonance research adopts a one-time centrifugal transfer method to avoid sample contamination or loss caused by manual transfer, and can ensure the uniformity of the biological sample in the nuclear magnetic rotor as much as possible, ensuring the accuracy of the nuclear magnetic test. Biological macromolecules need to be in a hydrated environment to fold correctly, and a hydrated environment can ensure the dynamic characteristics of the molecules, which is the key to function. If the sample is dry, the movement of the protein at the atomic level will be fixed and slowed down, affecting the function. The transfer method of the present application and the supporting centrifugal device can reduce the loss during the transfer process, maintain the water content of the sample to be tested (20%-50%), improve the resolution of the sample nuclear magnetic signal and maintain its corresponding biological functionality.

[0088] The transfer method is completed by cooperating with a centrifugal device, an ultracentrifuge sample tube 100, a nuclear magnetic rotor 200 and a centrifuge tube 300, and includes the following steps:

[0089] S1, preparation stage: enrich the sample to be tested into the ultracentrifuge sample tube 100. Specifically, the sample to be tested is firstly enriched into an ultracentrifuge sample tube 100 by centrifugation. Then the supernatant solution of the ultracentrifuge sample tube 100 is removed, and the water on the wall of the ultracentrifuge sample tube 100 is removed. The water content of the sample to be tested is maintained at 20% to 50%, and the sample sediment at the bottom of the tube is retained. Among them, the model or size of the ultracentrifuge sample tube (also called a centrifuge tube) can be selected in various ways. In this example, a round-bottom centrifuge tube is used.

[0090] S2, assembly stage: Place the nuclear magnetic rotor 200 into the tube slot 21 of the lower part 2 of the device, and then assemble the lower part 2 of the device, the upper part 1 of the device, and the ultracentrifuge sample tube 100 into the centrifuge tube 300 in sequence. Specifically, first place the nuclear magnetic rotor 200 without a cover into the tube slot 21 of the lower part 2 of the device, then place the upper part 1 and the lower part 2 of the device into the centrifuge tube 300 for assembly, and press the upper part 1 and the lower part 2 of the device. Then, the ultracentrifuge sample tube 100 with sample precipitation is inserted upside down into the confluence chamber 111 of the upper part 1 of the device. Of course, the ultracentrifuge sample tube 100 can also be inserted into the confluence chamber 111 of the upper part 1 of the device in an upright direction. In this case, a small opening needs to be opened at the bottom of the ultracentrifuge sample tube 100. This upright situation is generally used for conical bottom centrifuge tubes. The overall height of the device needs to be controlled to ensure that it does not exceed the height of the centrifuge tube 300. After loading, the distance from the tube mouth of the core tube is 5 to 15 mm to ensure the safety of the experimental operation.

[0091] S3, centrifugal transfer: The sample to be tested in the ultracentrifuge sample tube 100 is transferred to the nuclear magnetic rotor 200 through ultracentrifugation. Specifically, before centrifugation, the entire device needs to be weighed and balanced. Since the density of the material is relatively large, the direct method is to use the same material as the device to make a cylindrical structure with similar shape characteristics and put it into the centrifuge tube. The weight of the cylindrical structure and the device is close or slightly smaller, and the difference in total weight between the two can be made up by adding a certain amount of water to the balanced centrifuge tube. If there are two sets of identical devices, they can also be used together, one for sample loading and one for water addition for centrifugal balance. Through ultracentrifugation, the target sample will pass through the upper part 1 of the device and enter the nuclear magnetic rotor 200 placed in the lower part 2 of the device. Centrifugation chooses to use a horizontal centrifugal rotor, so that after a certain period of centrifugal compaction, the upper surface of the sample inside the nuclear magnetic rotor 200 will be parallel to the surface of the tube mouth, which is conducive to covering the tube cover. It should be noted that before loading the sample, you should have an understanding of the amount that the nuclear magnetic rotor 200 can load. The volume that a certain model of NMR rotor 200 can load is known. It is necessary to prepare an appropriate amount of sample to be tested to ensure that the sample volume is 75-80% of the volume of the NMR tube. Too much sample will overflow the NMR tube, and too little sample will not be fully rotated, which will cause difficulty in spinning the sample and too low a signal.

[0092] S4, capping step: take out the NMR rotor 200 filled with samples, select a suitable rotor cap and seal it tightly.

[0093] Example 4

[0094] Using the centrifugal device of Example 2 and the transfer method of Example 3

[0095] Taking human RIP3 fibrin (The structure of a minimum amyloid fibril coreformed by necroptosis-mediating RHIM of human RIPK3. Proc. Natl. Acad. Sci. USA 2021 118 (14) e2022933118) as an example, the transfer method and application effect of the centrifugal device of the present invention are described. S1. Purification and preparation of the obtained 13 C isotope labeled RIP3 protein, the protein is in a fibrous state, dispersed in the solution. Beckman Coulter Ultracentrifuge, optimaMAX-TL (rotor is Fixed Angle, TLA-55; centrifuge tube is Polypropylene, Snap-onCap, 1.5ml), centrifugation is used to enrich RIP3 fibrils, the speed is 55000rpm, centrifugation is carried out at 25℃ for 1 hour, and the amount of protein collected is about 15mg.

[0096] S2, centrifugal device as Figure 8 The assembly is described. Carefully remove the supernatant of the centrifuge tube (i.e., ultracentrifuge sample tube 100) of step S1, maintain the water content of RIP3 fibrin at 20% to 50%, and carefully absorb the droplets hanging on the tube wall with absorbent paper. The obtained RIP3 protein fibers remain at the bottom of the tube. Transfer the centrifuge tube containing RIP3 fibrin to the centrifuge device, press each part tightly, and place the whole in the centrifuge tube 300 (the centrifuge tube is polypropylene, open-top thin wall, 13.2ml).

[0097] S3. Use Beckman Coulter Ultracentrifuges, Optima XPN and matching rotor (Swinging Bucket, SW 41Ti) at 20,000 rpm and 4°C for centrifugation for 20 minutes.

[0098] S4. Take out the NMR rotor, and make sure the sample volume in the supernatant sample tube accounts for 75-80% of the volume of the NMR rotor. Finally, carefully cover the tube cap.

[0099] S5, the NMR rotor was placed in the NMR spectrometer for solid-state NMR sampling. The experiment was conducted on a Bruker 700MHz Neo device, using a 3.2mm MAS HCN probe, setting the temperature to 293K, and MAS = 15kHz. ) 13 C- 13 C Two-dimensional correlation spectrum (spectrum as shown Fig.10 , DARR mixing time 50ms). Simultaneously collect one-dimensional 1 H spectrum (spectrum as shown Fig. 9 ), monitor the water content in the sample. The resolution of the NMR signal of the sample with a high water content is high.

[0100] Example 5

[0101] The difference from Example 4 is that the sample is mouse RIPK3 fiber, the NMR test conditions are Bruker 700MHz Neo equipment, using a 3.2mm MAS HCN probe, setting the temperature to 303K, MAS = 15kHz. ) 13 C- 13 C two-dimensional correlation spectrum (spectrum as shown Fig.11 , DARR mixing time 50ms).

[0102] Comparative Example

[0103] The difference between the comparative example and Example 5 is that the centrifugal device of the present invention is not used, but a small medicine spoon is used to manually transfer to the nuclear magnetic rotor, and then the same test conditions as in Example 5 are used to obtain13 C- 13 C two-dimensional correlation spectrum, the spectrum is as follows Fig.12 .

[0104] from Fig.11 and Fig.12 It can be seen that a higher-resolution nuclear magnetic resonance spectrum can be obtained by using the transfer method and the matching centrifugal device of the present invention.

[0105] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions. The sentence "includes an element defined by ..." does not exclude the existence of other identical elements in the process, method, article or device including the element".

[0106] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A biological sample encapsulation and transfer method suitable for solid nuclear magnetic resonance research, wherein the transfer method is to transfer the sample in an ultracentrifuge sample tube (100) to a nuclear magnetic rotor (200) by a one-time centrifugation, and the transfer method is completed by using a centrifuge device, an ultracentrifuge sample tube (100), a nuclear magnetic rotor (200) and a centrifuge tube (300) in combination, and comprises the following steps: S1, enriching the sample to be tested into an ultracentrifuge sample tube (100); S2, placing the nuclear magnetic rotor (200) into the lower part (2) of the device, and then sequentially assembling the lower part (2), the upper part (1) of the device and the ultracentrifuge sample tube (100) into the centrifuge tube (300); S3, transferring the sample to be tested in the ultracentrifuged sample tube (100) to a nuclear magnetic rotor (200) through ultracentrifugation; The centrifugal device comprises an upper device part (1) and a lower device part (2), wherein the bottom of the upper device part (1) is detachably connected to the top of the lower device part (2); The upper part (1) of the device is provided with a flow guiding channel, and the flow guiding channel penetrates the upper part (1) of the device from top to bottom; The lower part (2) of the device is provided with a pipe placement groove (21) for placing the nuclear magnetic rotor (200); in, The head end of the flow guide channel is used for sample introduction, and the tail end is used for communication with the tube placement groove (21).

2. The transfer method according to claim 1, characterized in that: The upper part (1) of the device is provided with a flow converging portion (11) and a flow guiding portion (12), the flow converging portion (11) and the flow guiding portion (12) are respectively provided with a flow converging cavity (111) and a flow guiding cavity (121), the flow converging cavity (111) and the flow guiding cavity (121) are connected to form the flow guiding channel; The inner diameter of the converging cavity (111) is greater than the inner diameter of the guiding cavity (121).

3. The transfer method according to claim 2, characterized in that: The inner diameter of the flow guiding cavity (121) decreases from top to bottom; And / or, the flow converging portion (11) and the flow guiding portion (12) are both hollow to form the flow converging cavity (111) and the flow guiding cavity (121); And / or, the converging portion (11) is used to place the ultracentrifuge sample tube (100), and the shape of the converging portion (11) is adapted to the shape of the sample outlet of the ultracentrifuge sample tube (100).

4. The transfer method according to claim 1, characterized in that: A boss (13) is provided at the bottom of the upper part (1) of the device, a groove (22) adapted to the boss (13) is provided at the top of the lower part (2) of the device, and the groove (22) is located at the top of the tube placement groove (21) and is in communication with the tube placement groove (21); The guide channel passes through the boss (13), and the center line of the guide channel coincides with the center line of the tube placement groove (21).

5. The transfer method according to claim 4, characterized in that: The boss (13) comprises a connecting section (131) and a hollow extending section (132); the connecting section (131) is adapted in shape to the groove (22), and the outer diameter of the hollow extending section (132) is smaller than the inner diameter of the tube placement groove (21); And / or, a sealing ring is provided on the outer side of the boss (13) or the inner side of the groove (22); Alternatively, the boss (13) is threadedly connected to the groove (22).

6. The transfer method according to claim 5, characterized in that: The difference between the length of the connecting section (131) and the depth of the groove (22) is 0 to 1 mm; And / or, the length of the hollow extension section (132) is 1 to 3 mm.

7. The transfer method according to any one of claims 1 to 6, characterized in that: The centrifugal device is made of a material that meets the requirements of high-speed centrifugation; And / or, the upper part (1) of the device is cylindrical, the lower part (2) of the device is cylindrical with a hemispherical bottom, and the outer diameters of the upper part (1) and the lower part (2) of the device are evenly matched with the inner diameter of the centrifuge tube; And / or, the centrifugal device can be manufactured by precision machine tool processing or 3D printing.

8. The transfer method according to claim 1, characterized in that: The sample to be tested is enriched into an ultracentrifuge sample tube (100) by centrifugation, wherein the water content of the sample to be tested is 20% to 50%; and / or, the volume of the sample to be tested in the ultracentrifuge sample tube (100) is 3 / 4 to 4 / 5 of the volume of the nuclear magnetic rotor (200); and / or, in step S3, the centrifuge rotor used in the ultracentrifugation process is placed horizontally; And / or, the working conditions of the ultracentrifugation are: centrifugal speed of 15000-25000 rpm, working temperature of 3-5°C, and centrifugal time of 10-30 min.

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